Electron transport is the general process in cells by which electrons generated from the oxidation of molecules such as NADH and FADH2 are transferred, through the action of various enzymes, to a series of electron carriers. These electron carriers may act as electron donors themselves for various reductive reactions in the cell or may transport their electrons to other electron carriers along an electron transport chain. The change in oxidation potential as electrons are passed along such a chain generates energy which may be used by the cell.
The mitochondrial electron transport (or respiratory) chain is a series of enzyme complexes in the mitochondrial membrane responsible for the transport of electrons from NADH through a series of redox centers (electron carriers) within these complexes to oxygen and for the coupling of this oxidation to the synthesis of ATP (oxidative phosphorylation). ATP provides the primary source of energy for driving a cell's many energy-requiring reactions.
Most electron carriers are prosthetic groups, such as flavins, heme, iron-sulfur clusters and copper, bound to protein particles. Ubiquinone (Coenzyme Q) is the only electron carrier that is not protein bound. The cytochromes (Cyts) are one type of electron carrier protein; cytochromes are related to one another by the presence of a bound heme group consisting of a porphyrin ring containing a tightly bound iron atom. The iron atom serves as the actual electron carrier by changing from the ferric to the ferrous state when accepting an electron. Iron-sulfur proteins are a second major family of electron carriers in which either two or four iron atoms are bound to sulfur atoms and to cysteine side chains forming an iron-sulfur center. Ubiquinone, the simplest of electron carriers, includes a quinone ring attached to a hydrophobic tail which anchors it to the mitochondrial membrane. In addition to six different heme-linked cytochromes, more than six iron-sulfur centers, and ubiquinone, there are also two copper atoms and a flavin (FMN) serving as electron carriers in the pathway from NADH to oxygen.
The key enzyme complexes in the respiratory chain are NADH:ubiquinone oxidoreductase (NADH-D), succinate:ubiquinone oxidoreductase, cytochrome c1-b oxidoreductase, cytochrome c oxidase (COX), and ATP synthase. All of these complexes are located on the inner matrix side of the mitochondrial membrane except succinate:ubiquinone oxidoreductase, which is located on the cytosolic side. NADH-D accomplishes the first step in the respiratory chain by accepting electrons from NADH and passing them through a flavin molecule and several iron-sulfur centers to ubiquinone. Succinate:ubiquinone oxidoreductase also transports electrons generated by oxidation of succinate to fumarate in the citric acid cycle through electron carriers (FAD and iron-sulfur centers) to the membrane bound ubiquinone. Cytochrome c1-b oxidoreductase accepts electrons from ubiquinone and passes them on to cytochrome c. COX accepts electrons from cytochrome c and catalyzes the last, and most important, transfer of electrons to oxygen. Energy released in the course of each of these electron transfers is harnessed by ATP synthase to form ATP (oxidative, phosphorylation).
NADH-D, the largest of these complexes with an estimated mass of 800 kDa, contains some 40 polypeptide subunits of widely varying size and composition. The polypeptide composition of NADH-D is similar in a variety of mammalian species including rat, rabbit, cow, and human (Cleeter, M. W. J. and Ragan, C. I. (1985) Biochem. J. 230: 739-46). The best characterized NADH-D is from bovine heart mitochondria and is composed of 41 polypeptides (Walker, J. E. et al. (1992) J. Mol. Biol. 226: 1051-72). Seven of these polypeptides are encoded by mitochondrial DNA, while the remaining 34 are nuclear gene products that are imported into the mitochondria. Six of these imported polypeptides are characterized by N-terminal signal peptide sequences which target these polypeptides to the mitochondria and are then cleaved from the mature proteins. A second group of polypeptides lack N-terminal targeting sequences and appear to contain import signals which lie within the mature protein (Walker et al., supra). The measured molecular masses of several of the smaller polypeptides, B8, B13, B14, B15, and B22, are consistent with post-translational removal of the terminal methionine residue and N-acetylation of the adjacent amino acid.
The functions of many of the individual subunits in NADH-D are largely unknown. The 24-, 51-, and 75-kDa subunits have been identified as being catalytically important in electron transport, with the 51-kDa subunit forming part of the NADH binding site and containing the flavin moiety that is the initial electron acceptor (Ali, S. T. et al. (1993) Genomics 18:435-39). The location of other functionally important groups, such as the electron-carrying iron-sulfate centers, remains to be determined. Many of the smaller subunits (<30 kDa) contain hydrophobic sequences that may be folded into membrane spanning α-helices. These subunits presumably are anchored into the inner membrane of the mitochondria and interact via more hydrophilic parts of their sequence with globular proteins in the large extrinsic domain of NADH-D.
COX is composed of thirteen polypeptide subunits, three of which are mitochondrial gene products, and the ten remaining subunits of which are nuclear gene products (Lomax, M. I. et al. (1990) Gene 86: 209-16). The catalytic and protein-transducing functions and the site of interaction with cytochrome c are all associated with the mitochondrial, gene products, subunits 1 through 3. The exact functions of the ten smaller, nuclear-encoded subunits, 4 through 13, are unknown, but it has been suggested that they regulate oxidative energy output (Lomax et al., supra).
NADH cytochrome b5 reductase is an enzyme that specifically serves to oxidize the electron carrier cytochrome b5 which, in turn, becomes a central electron donor for various reductive reactions occurring on the cytoplasmic surface of liver endoplasmic reticulum (Strittmatter, P. et al. (1992) J. Biol. Chem. 267: 2519-23). Both cytochrome b5 reductase, and cytochrome b5 are amphipathic molecules composed of globular hydrophilic catalytic domains linked through short flexible sequences to membrane-anchoring hydrophobic domains that serve to orient the catalytic sites at the membrane-aqueous interface and permit rapid electron transfer. Three lysine residues in cytochrome b5 reductase, K41, K125, and K163, are implicated in the formation of charged ion pairs with carboxyl groups on cytochrome b5 during interactions between the active sites of the two proteins (Strittmatter, P. et al. (1990) J. Biol. Chem. 265: 21709-13). Site-directed mutagenesis studies demonstrate marked decreases in catalytic efficiency when any of these three lysine residues are replaced by negatively charged amino acids (Strittmatter et al. (1992), supra).
Defects and altered expression of NADH-D are associated with a variety of human diseases, including neurodegenerative diseases, myopathies, and cancer (Singer, T. P. et al. (1995) Biochim. Biophys. Acta 1271:211-19; Selvanayagam, P. and Rajaraman, S. (1996) Lab. Invest. 74:592-99). In addition, NADH-D reduction of the quinone moiety in chemotherapeutic agents such as doxorubicin is believed to contribute to the antitumor activity and/or mutagenicity of these drugs (Akman, S. A. et al. (1992) Biochemistry 31:3500-6).
The discovery of new electron transport proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cancer, immune disorders, and reproductive disorders.